Data on the clinical presentation, risk factors, and outcomes for healthcare personnel (HCP) infected with influenza A H1N1 virus (H1N1) are limited. From June to July 2009, a prospective study was conducted among HCP with influenza-like illness (ILI) at university hospitals in Buenos Aires. A reverse transcription polymerase chain reaction (RT-PCR) was used to diagnose H1N1. A logistic regression model was developed to identify factors associated with H1N1. Among 1519 HCP, 96 (6.3%) were diagnosed with an ILI. Of these, 85 (88.5%) were swabbed for H1N1 detection, with 43 positive cases (2.8%). Seasonal influenza immunization was recorded in 76%. Comparison of H1N1-positive vs. H1N1-negative cases showed that H1N1-positive cases more frequently had asthenia (72% vs. 48%, p = 0.03) and cough (79% vs. 43%, p = 0.008) and less frequently had diarrhoea (9% vs. 29%, p = 0.03) and prior prophylaxis with oseltamivir (5% vs. 31%, p = 0.002). The logistic regression model showed that presence of cough (odds ratio (OR) 6.93, 95% confidence interval (CI) 2.24, 21.4) was associated with an increased risk of H1N1. Prior prophylaxis with oseltamivir (OR 0.08, 95% CI 0.01, 0.43) was associated with a lower probability of H1N1 infection. A high proportion of HCP with an ILI were infected with H1N1. Complication rates were relatively low. Prior prophylaxis with oseltamivir was associated with a lower risk of developing H1N1.