Bile acid homeostasis plays a critical role in regulating human physiology and influences disease outcomes. The bile acid pool is generated in the liver and is modified in the gastrointestinal tract by multiple gut microbial enzymes, starting with bile salt hydrolases (BSHs) that deconjugate amino acids from the sterol core. Recent studies have shown that some BSHs can also re-conjugate bile acids with various amino acids producing microbially conjugated bile acids (MCBAs), which also serve as substrates for BSH-mediated deconjugation. BSH enzymes from Lactobacillaceae family and a small number of other gut microbial taxa have been examined structurally and biochemically, but the full scope of BSH substrate specificities, including for the MCBAs, is still unclear. Here, we interrogate a diverse panel of BSHs from the human gut microbiome for their deconjugation activities of several substrates including MCBAs. We also evaluated the efficacy of three BSH inhibitors against several BSHs and examined the binding mode of each inhibitor via x-ray crystallography. The inhibitor studies informed the development of three novel activity-based BSH probes, one of which is a promising candidate for subsequent chemoproteomics investigations of BSHs in human fecal samples. Our findings advance our understanding of the structural features present in diverse gut microbial BSH enzymes toward elucidating the roles these non-host factors play in host physiology, homeostasis, and transitions to disease.