Post-COVID-19 pandemic, most universities changed their educational model from online courses to onsite learning, allowing students to attend regular face-to-face classes. These changes can cause stress in students, which affects physical fitness. The aim of this study was to investigate the relationship between stress levels and physical fitness in female university students. The participants were 101 female university students, 18–23 years of age. All participants completed the Suan Prung Stress Test-60 (SPST-60). The physical fitness test included body composition, cardiorespiratory fitness, as well as musculoskeletal fitness. Multiple linear regression analysis was used to determine the associations between SPST-60 scores and physical fitness. A p-value < 0.05 was considered statistically significant. We found a negative correlation between the sources of stress scores, here environment, and maximal oxygen consumption (β = −0.291; 95% CI, −0.551, −0.031). We also found that symptoms of stress scores in the parasympathetic and sympathetic nervous systems were positively associated with waist-hip circumference ratio (WHR) (β = 0.010; 95% CI, 0.002, 0.017 and β = 0.006; 95% CI, 0.000, 0.012, respectively). Moreover, the symptoms of stress, here emotion, were positively associated with the WHR (β = 0.005; 95 %CI, 0.001, 0.009) and negatively associated with upper extremity muscle strength (β = −0.005; 95% CI, −0.009, 0.000). The results of this study confirmed the associations between stress levels in the post-COVID-19 pandemic era and WHR, maximal oxygen consumption, and upper extremity muscle strength. As a result, stress reduction or prevention alternatives should be considered in order to maintain physical fitness and prevent stress disorders.