Trehalose-6-phosphate phosphatase (TPP), a key enzyme in bacterial trehalose biosynthesis, is essential for the stress response and represents a promising target for antimicrobial development. In this study, we performed a comparative functional analysis of TPP homologs from three bacterial species—Stenotrophomonas maltophilia (St-TPP), Xanthomonas axonopodis (Xa-TPP), and Dermatophilus congolensis (Dc-TPP)—complemented by structural characterization of a representative enzyme. Phosphatase activity assays revealed notable differences in catalytic efficiency (kcat/Km) among TPP homologs, with approximately fivefold variation. Dc-TPP exhibited higher substrate affinity (Km of 0.087 mM), whereas St-TPP showed higher catalytic turnover (kcat of 0.87 s⁻¹). The measured kinetic parameters fall toward the lower end of the range reported for bacterial TPPs, which typically span several orders of magnitude (~10¹–10⁴ M⁻¹·s⁻¹), while relative differences among the tested homologs were consistently observed under identical assay conditions. To provide structural context for these observations, we determined the 2.5 Å crystal structure of the apo form of Dc-TPP. The structure reveals a conserved HAD-like catalytic core and a cap domain positioned above the active site. In the absence of ligand, the cap domain adopts a conformation that partially occludes the catalytic pocket, which may be consistent with a pre-catalytic or gated state. Although catalytic residues are highly conserved among TPP homologs, the observed functional differences may be consistent with variations in cap-domain orientation and dynamics, which could influence substrate accessibility and catalytic turnover. These findings provide a structure–function framework for TPP and highlight cap-domain mobility as a potential modulator of enzymatic activity, with implications for rational inhibitor design.