Bacteria are ubiquitous in the environment and critical to human health and disease, yet only a small fraction can be identified through standard culture methods. Advances in next-generation sequencing techniques have improved bacterial identification, but these DNA-based methods cannot distinguish live bacteria from relic DNA. Recently, DNA-labeling dyes (e.g., 5-bromo-2'-deoxyuridine [BrdU] and propidium monoazide [PMA]) have been used to detect metabolically active bacteria in different sample types. Here, we compare BrdU and PMA in combination with 16SrRNA gene sequencing to characterize metabolically active bacteria in two different sample types: (1) manufactured products ( = 78; cigarettes, hookah, and little cigar) and (2) natural samples ( = 186; rainwater, soil, and produce). Metabolically active bacterial communities identified in BrdU-labeled samples had lower alpha diversity than that of PMA-treated and non-treated samples. , , , and were observed in all the samples tested. Irrespective of sample type, was predominant in BrdU-treated samples, while was more abundant in non-treated samples compared to PMA-treated samples. We also observed that PMA-treated samples tend to overestimate the metabolically active bacterial fraction compared to BrdU-treated samples. Overall, our study highlights how different labeling techniques influence bacterial community analysis findings, underscoring the need for careful selection of labeling approaches when assessing environmental samples.