Literature shows that music can reduce stress conditions. This pilot study investigated the effects of music listening on work-related stress and well-being in healthcare professionals. A total of 45 subjects were randomly assigned to three treatment groups: No Music, Individualized Music and Melomics-Health Listening. Music groups experienced a daily 30-min-playlist listening for 3 weeks at home. The Maugeri Stress Index-Revised (MASI-R) and the Psychological General Well-Being Index (PGWBI) were administered at baseline, after 3 weeks and after 7 weeks (follow-up). Longitudinal data were analyzed by means of a nested ANOVA model, testing the main effects of time and treatment and the interaction between them. MASI-R scores showed a positive trend in music groups and a worsening in the control group. Only the interaction time/treatment emerged as supporting a trend toward statistical significance (P = 0.07). PGWBI showed a stability in music groups and a clear decline in controls, without significant effects. Results from the study support the need for a larger clinical trial: it is suggested that daily music listening could be implemented to reduce work-related stress and that the effects may be related, not only to individual musical preferences and familiarity, but also to specific music structures and parameters.