The COVID-19 outbreak has led to relevant changes in everyday life worldwide. One of these changes has been a rapid transition to and an increasing implementation of working from home (WH) modality. This study aimed to evaluate the impact of mandatory WH during the COVID-19 pandemic on lifestyle behaviors, Mediterranean diet adherence, body weight, and depression. An online cross-sectional survey was conducted in the early 2022 at the National Research Council of Italy using ad hoc questions and validated scales collecting information on physical activity, sedentary behavior, hobbies/pastimes, dietary habits including adherence to the Mediterranean diet, body weight, and depression during WH compared with before WH. 748 respondents were included in the study. An increased sedentary lifetime was reported by 48% of respondents; however, the subsample of workers who previously performed moderate physical activity intensified this activity. Body weight gain during WH was self-reported in 39.9% of respondents. Mediterranean diet adherence increased (p≪0.001) during WH compared with before WH. The average level of mental health did not record an overall variation; however, the proportion of subjects with mild and moderate depression increased (p = 0.006), while workers who reported values indicative of depression before the transition declared an improvement. These findings highlight health-related impact of WH during the COVID-19 pandemic that may inform future strategies and policies to improve employees’ health and well-being.