This work addresses in vitro the potential impact of two food additives in the safety spotlight, i.e., the flavor enhancer monosodium glutamate (MSG) and the azo-dye colorant allura red AC (AR), on the human gut microbiota from healthy donors by using in vitro faecal batch culture models, and an intestinal cell line model. The rationale to investigate MSG and AR safety on the gut microbiota is motivated by recent research pointing to a contribution of their chronic consumption to intestinal inflammation. The short-term exposure of faecal samples to the individual additives at two single doses (0.8 and 0.08 mM for MSG; and 0.6 and 0.06 mM for AR) did not exert significant changes in the relative abundance of gut bacteria as determined through 16S rRNA sequencing. MSG and AR were degraded into γ-aminobutyric acid (GABA) and 4-amino-5-methoxy-2-methylbenzenesulfonic acid (4A5M2M), respectively, during fermentation. While GABA was exclusively derived from microbial activity, a partial degradation of AR to 4A5M2M was triggered by heat treatment of AR-containing samples prior to faecal fermentation. Further, MSG and AR alone or after exposure to faecal samples did not show cytotoxic effects on intestinal HT29 cell monolayers. These results highlight the role of the human gut bacteria and the impact of heat-based processing in the bioconversion of certain food additives. Our findings reinforce the safety of both food additives regarding their impact on the gut microbial communities under conditions resembling acute short-term exposures, although the potential biological effects of the metabolites generated from their degradation or from chronic exposures should be further investigated.