Electrospun polyamide (PA) nanofibers have great potential for medical applications (in dermatology as antimicrobial compound carriers or surgical sutures). However, little is known about microbial colonization on these materials. Suitable methods need to be chosen and optimized for the analysis of biofilms formed on nanofibers and the influence of their morphology on biofilm formation. We analyzed 11 PA nanomaterials, both nonfunctionalized and functionalized with AgNO, and tested the formation of a biofilm by clinically relevant bacteria ( CCM 4517, CCM 3953, and CCM 4418). By four different methods, it was confirmed that all of these bacteria attached to the PAs and formed biofilms; however, it was found that the selected method can influence the outcomes. For studying biofilms formed by the selected bacteria, scanning electron microscopy, resazurin staining, and colony-forming unit enumeration provided appropriate and comparable results. The values obtained by crystal violet (CV) staining were misleading due to the binding of the CV dye to the PA structure. In addition, the effect of nanofiber morphology parameters (fiber diameter and air permeability) and AgNO functionalization significantly influenced biofilm maturation. Furthermore, the correlations between air permeability and surface density and fiber diameter were revealed. Based on the statistical analysis, fiber diameter was confirmed as a crucial factor influencing biofilm formation ( ≤ 0.01). The functionalization of PAs with AgNO (from 0.1 wt %) effectively suppressed biofilm formation. The PA functionalized with a concentration of 0.1 wt % AgNO influenced the biofilm equally as nonfunctionalized PA 8% 2 g/m. Therefore, biofilm formation could be affected by the above-mentioned morphology parameters, and ultimately, the risk of infections from contaminated medical devices could be reduced.