Introduction: The term “WORKbiota” has been used to describe the impact of occupational exposure and work types on human microbiota composition. Airline pilots, construction workers, and fitness instructors encompass three diverse professional groups, each with distinct work environments and lifestyle factors that may significantly influence their intestinal “WORKbiota.”Objectives: The current preliminary investigation was aimed to compare the relative abundance of specific gut microbes among airline pilots, construction workers, and fitness instructors to shed light on any significant differences. By scrutinizing these diverse professional groups, our objective was to enhance our understanding of how occupational factors influence gut microbiota while identifying possible implications for occupational medicine.Methods: A convenience sample consisting of 60 men representing three different professional domains - airline pilots, construction workers, and fitness instructors (with 20 individuals in each group) - was selected during regular outpatient occupational health consultations. The abundance of selected gut microbiota constituents, includingEscherichia coli,Methanobrevibacter smithii,Akkermansia muciniphila,Faecalibacterium prausnitzii,Lactobacillusspp.,Bifidobacteriumspp., andBacteroidesspp., was quantified using quantitative SYBR Green quantitative real-time polymerase chain reaction (qRT-PCR) in stool samples.Results: There were no significant variations among the groups concerningEscherichia coli,Methanobrevibacter smithii,Bifidobacteriumspp., andBacteroidesspp. However,Lactobacillusspp. andFaecalibacterium prausnitziiwere significantly more abundant in the microbiota of fitness instructors compared to both airline pilots and construction workers, with no significant differences observed between the latter two groups. Notably, the abundance ofAkkermansia muciniphilademonstrated a progressive decline from fitness instructors to construction workers and ultimately to airline pilots, who exhibited the lowest levels.Conclusion: Airline pilots’ gut microbiota was characterized by a lower abundance of health-promoting bacterial species, includingLactobacillusspp.,Faecalibacterium prausnitzii, andAkkermansia muciniphila. Future research is essential to determine whether targeted interventions, such as probiotic and prebiotic supplementation, could potentially enhance gut microbiota composition and overall health in particular occupational groups.