During the lockdown for the coronavirus disease 2019 (COVID-19), entire populations were instructed to live in home confinement. We investigated the effects of the COVID-19 lockdown on the physical activity (PA) and mental health of students and employees in a Colombian University. A cross-sectional study was conducted through an online survey during the first isolation. A total of 431 respondents (192 males) aged 18–60 years old (28.1 ± 11.1 years) participated. The international Physical Activity Questionnaire (IPAQ) and the short version of the Psychological General Well-Being Index (PGWBI-S) were used. The lockdown had a negative effect on PA levels, with students exhibiting the greatest decrease (~34%; p ˂ 0.001) compared to employees (~24%; p ˂ 0.01). The analysis showed a greater change in PA behavior before and during the lockdown in highly active student participants (5750 vs. 5141 MET min/week; p < 0.05). Additionally, the psychological assessment revealed a lower score in students compared to employees in the male (70.1 vs. 82.6) and female groups (60.2 vs. 79.6). Moreover, the results revealed an influence of sex, with only the female students exhibiting a state of distress. Self-reported PA and psychological well-being were compromised during the COVID-19 lockdown in the academic community, with students and females being more affected.