Previous studies have characterized bacterial communities in menthol versus non-menthol cigarettes. However, these studies evaluated commercial cigarettes, for which levels of chemical constituents are largely unknown, and therefore, could not assess the impact of varying nicotine and menthol concentrations on tobacco bacterial communities. To address this knowledge gap, we performed time-series experiments using SPECTRUM research cigarettes with varying nicotine and menthol levels. Cigarettes were incubated under three storage conditions for 14 days. Cigarette tobacco was then sub-sampled (n = 288), DNA extracted, and subjected to PCR amplification of the V3V4 region of the 16S rRNA gene, followed by Illumina HiSeq sequencing. Sequences were analyzed using QIIME and R. Incubation under varying conditions did not affect bacterial diversity. However, significant differences in bacterial communities were observed across varying nicotine concentrations in menthol and non-menthol products. For example, Pseudomonas spp. was negatively correlated with nicotine concentrations in menthol cigarettes. A significantly higher relative abundance of P. veronii and P. viridiflava was observed in menthols versus non-menthols, while a significantly higher relative abundance of Bacillus foraminis and B. coagulans was found in non-menthols versus menthols. Additional bacteria (e.g., Staphylococcus spp., Jeotgalicoccus psychrophilus, and B. flexus) significantly changed in relative abundance between days 0 and 14. Our findings demonstrate that nicotine and menthol levels have a significant impact on the relative abundance of potential bacterial pathogens present in cigarettes. Future work is needed to demonstrate whether these tobacco-associated bacteria could be transferred to users while smoking, ultimately contributing to adverse respiratory impacts.