This cross-sectional study investigated knowledge, attitudes, and practices (KAP) regarding bovine tuberculosis surveillance and analyzed factors influencing KAP among Thai slaughterhouse personnel, utilizing a One Health approach. A validated questionnaire was administered to 208 participants across five geographical regions of Thailand. Statistical analysis revealed moderate mean scores for knowledge (5.28/10), attitudes (38.55/65), and practices (34.62/50). Significant differences were observed in knowledge scores across education levels (F = 3.427, p = 0.005) and job positions (F = 4.562, p = 0.011), with higher education and managerial positions being associated with better performance. Path analysis demonstrated positive correlations among KAP components (p < 0.05), with the strongest correlation between attitudes and practices (r = 0.543). The most significant risk factors for poor practices were identified as lack of training (OR = 2.76, 95% CI: 1.45–5.24, p = 0.002) and inadequate tuberculosis screening (OR = 2.31, 95% CI: 1.18–4.52, p = 0.015). The Knowledge–Practice gap index of −31.14 indicated substantial discrepancy between knowledge and implementation. Developing targeted training programs is needed by focusing on knowledge enhancement and awareness-building, as well as fostering inter-agency collaboration in line with the One Health approach enhance the efficiency of bovine tuberculosis surveillance in Thailand.
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