Arylamines constitute a large group of industrial chemicals detoxified by certain bacteria through conjugation reactions catalyzed by -acetyltransferase (NAT) enzymes. NAT homologs, mostly from pathogenic bacteria, have been the subject of individual studies that do not lend themselves to direct comparisons. By implementing a practicable pipeline, we carried out a comparative investigation of 15 NAT homologs from 10 bacteria, mainly bacilli, streptomycetes, and one alphaproteobacterium. The new homologs were characterized for their sequence, phylogeny, predicted structural features, substrate specificity, thermal stability, and interaction with components of the enzymatic reaction. NATs demonstrated the characteristics of xenobiotic metabolizing -acetyltransferases, with the majority of homologs generating high activities. Nonpathogenic bacilli are thus proposed as suitable mediators of arylamine bioremediation. Of the homologs, the NAT2 isoenzyme of efficiently transformed highly toxic arylamines, while the remaining homologs were inactive or generated low activities, suggesting that xenobiotic metabolism may not be their primary role. The functional divergence of NATs was consistent with their observed sequence, phylogenetic, and structural variability. These and previous findings support classification of microbial NATs into three groups. The first includes xenobiotic metabolizing enzymes with dual acetyl/propionyl coenzyme A (CoA) selectivity. Homologs of the second group are more rarely encountered, acting as malonyltransferases mediating specialized ecological interactions. Homologs of the third group effectively lack acyltransferase activity, and their study may represent an interesting research area. Comparative NAT enzyme screens from a broad microbial spectrum may guide rational selection of homologs likely to share similar biological functions, allowing their combined investigation and use in biotechnological applications. Arylamines are encountered as industrial chemicals or by-products of agrochemicals that may constitute highly toxic contaminants of soils and groundwaters. Although such chemicals may be recalcitrant to biotransformation, they can be enzymatically converted into less toxic forms by some bacteria. Therefore, exploitation of the arylamine detoxification capabilities of microorganisms is investigated as an effective approach for bioremediation. Among microbial biotransformations of arylamines, enzymatic conjugation reactions have been reported, including NAT-mediated acetylation. Comparative investigations of NAT enzymes across a range of microorganisms can be laborious and expensive, so here we present a streamlined methodology for implementing such work. We compared 15 NAT homologs from nonpathogenic, free-living bacteria of potential biotechnological utility, mainly , which are known for their rich secondary and xenobiotic metabolism. The analysis allowed insights into the evolutionary and functional divergence of bacterial NAT homologs, combined with assessment of their fundamental structural and enzymatic differences and similarities.