Testosterone deficiency (TD) is a prevalent disorder in men, associated with a range of health complications. Dietary live microbe intake has garnered attention for its potential to modulate gut microbiota and promote human health. This study aims to investigate the association between dietary live microbe intake and the risk of TD in a large, nationally representative sample. We analyzed data from the National Health and Nutrition Examination Survey (NHANES) for the years 2013-2016. Dietary live microbe intake was estimated using a 24-hour dietary recall, and participants were categorized into low, medium, and high intake groups. TD was defined as a serum testosterone level below 300 ng/dL. Multivariable weighted logistic regression models were used to assess the association between different dietary live microbe intake and TD risk, adjusting for potential confounders. A total of 4,034 male participants were included in the analysis. High dietary live microbe intake was associated with a significantly lower risk of TD in all models. In the fully adjusted model (Model 3), the odds ratio (OR) for TD in the high intake group was 0.71 (95% CI: 0.53 to 0.96; p = 0.03) compared to the low intake group. Subgroup analyses showed consistent results across different population characteristics, particularly among those without diabetes (OR: 0.64; 95% CI: 0.47 to 0.88; p = 0.01) and without cardiovascular disease (OR: 0.64; 95% CI: 0.47 to 0.88; p = 0.02). Restricted Cubic Spline analysis revealed a linear inverse relationship between food intake and TD risk in the medium and high intake groups. Our findings suggest that a higher intake of dietary live microbes is associated with a reduced risk of TD, highlighting the potential of dietary modifications as a preventive strategy for TD. Further research, including longitudinal studies and clinical trials, is necessary to confirm these results and explore the underlying mechanisms.