Healthcare workers (HCWs) are frequently exposed to a variety of chemical agents, which can result in the development of allergic or irritant contact dermatitis. The present study aimed to assess the prevalence of skin sensitization among HCWs who presented with symptoms of contact dermatitis, considering both occupational and non-occupational risk factors. The study population comprised 127 HCWs who attended routine occupational health surveillance at the Tor Vergata Teaching Hospital in Rome between November 2023 and May 2024. A structured dermatitis questionnaire and patch testing were administered to the participants. Demographic and lifestyle data, including information on occupation, night shift work, smoking habits, and body mass index (BMI), were collected. Patch test positivity was observed in 31.5% of participants, with the most common clinical presentation being erythematous-desquamative allergic contact dermatitis. A significantly higher likelihood of patch test positivity was observed among nurses (57.1%), particularly for nickel sensitization, compared to other occupational groups. A trend towards an association between night shift work and skin sensitization was observed, although this did not reach statistical significance. No significant associations were found for ages over 35 years, sex, or BMI. These findings highlight the elevated risk of contact sensitization among nurses, emphasizing the need for targeted interventions, including exposure reduction strategies and protective measures, to mitigate occupational skin hazards in healthcare settings.