Purpose: This cross-sectional study examined the associations between health-related physical fitness (HPF) and cardiovascular disease (CVD) risk factors in overweight and obese university staff. Methods: A total of 340 university staff (109 women, mean age 43.1 ± 9.7years) with overweight (n = 284) and obesity (n = 56) were included. The HPF indicators included skeletal muscle mass index (SMI), body fat percentage (BFP), grip strength (GS), sit-and-reach test (SRT), and vital capacity index (VCI). CVD risk factors were measured, including uric acid (UA), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and glucose (GLU). Results: BFP, SMI, and GS were positively associated with UA level (β = 0.239, β = 0.159, β = 0.139, p < 0.05). BFP was positively associated with TG and TG/HDL-C levels (β = 0.421, β = 0.259, p < 0.05). GS was positively associated with HDL-C level (β = 0.244, p < 0.05). SRT was negatively associated with GLU level (β = −0.130, p < 0.05). Conclusions: In overweight and obese university staff, body composition, muscle strength, and flexibility were associated with CVD risk factors. An HPF test may be a practical nonmedical method to assess CVD risk.