Background At the beginning of the COVID-19 pandemic, some workers had the opportunity to work from home, while others remained in on-site work. The aim of the present study was to compare the psychosocial aspects, work ability, mental health conditions and infection rates of Brazilian workers in remote and on-site work through a longitudinal study with quarterly follow-up assessments over a 12-month period.Method A total of 1,211 workers from different economic sectors participated in the study, 897 of whom (74.1%) worked from home and 314 (25.9%) remained in on-site work. Psychosocial aspects were assessed using the Copenhagen Psychosocial Questionnaire (COPSOQ). Work ability was assessed using the Work Ability Index (WAI) and the Work Ability Score (WAS). Mental health conditions and infection rate were recorded based on self-reported medical diagnoses. Online questionnaires were answered from June 2020 to September 2021, involving two waves of the COVID-19 pandemic. The groups were compared using chi-square tests, t-tests, and two-way ANOVA.Results In the first wave of the pandemic, remote workers reported more quantitative demands and work-family conflicts, whereas on-site workers reported more emotional demands, low development of new skills, low commitment, low predictability, low recognition, and low satisfaction. They also reported greater occurrences of unwanted sexual attention, threats of violence, and physical violence. In the second wave, the remote group continued to report high work-family conflicts, whereas the on-site group reported – in addition to the results of the 1st wave – low influence at work, low quality of leadership, and burnout. No significant difference was found between groups with regards to the WAI in either wave (1st wave: P = 0.46; 2nd wave: P = 0.62). Most workers in both groups reported good work ability in both periods. For the WAS, a significant difference was found between the 3rd and 12th months (P < 0.01) in both groups, with a mean reduction of 0.4 points.Conclusions Psychosocial critical aspects differed between groups. Work ability and mental health conditions were similar between remote and on-site workers. The encouragement of remote work could have prevented COVID contamination in the Brazilian population.