Lifestyle factors such as smoking, sedentarism, low physical activity levels, and overweight are associated with poor health, and they can potentially influence work ability. However, it remains unknown which lifestyle habits are associated with work ability among physical therapists (PTs). The aim of this study was to examine the associations between smoking, alcohol consumption, BMI, sitting time, and physical activity levels with work ability among PTs utilizing a nationwide questionnaire. Associations were modeled using logistic regression controlled for various confounders. Overweight, sitting >150 min/day, and <75 min/week of leisure-time vigorous physical activity were associated with lower work ability among PTs. Further, the existence of two unhealthy habits showed a weak-to-moderate positive association with lower work ability scores (Model 1: OR, 2.21, 95% CI = 1.16–4.22; Model 2: OR, 2.32, 95% CI, 1.18–4.54), with even stronger associations when three unhealthy habits (Model 1: OR = 3.30, 95% CI, 1.58–6.86; Model 2: OR, 3.34, 95% CI, 1.54–7.26) or four unhealthy habits (Model 1: OR = 8.91, 95% CI, 2.55–31.1; Model 2: OR = 8.20, 95% CI, 2.15–31.2) were present. In conclusion, overweight, low physical activity, and sedentarism were associated with lower levels of work ability, especially when ≥2 unhealthy lifestyle factors were present.