Background. Melioidosis results from infection with Burkholderia pseudomallei and is associated with case-fatality rates up to 40%. Early diagnosis and treatment with appropriate antimicrobials can improve survival rates. Fatal and nonfatal melioidosis cases were identified in Puerto Rico in 2010 and 2012, respectively, which prompted contact investigations to identify risk factors for infection and evaluate endemicity. Methods. Questionnaires were administered and serum specimens were collected from coworkers, neighborhood contacts within 250 m of both patients' residences, and injection drug user (IDU) contacts of the 2012 patient. Serum specimens were tested for evidence of prior exposure to B. pseudomallei by indirect hemagglutination assay. Neighborhood seropositivity results guided soil sampling to isolate B. pseudomallei. Results. Serum specimens were collected from contacts of the 2010 (n = 51) and 2012 (n = 60) patients, respectively. No coworkers had detectable anti–B. pseudomallei antibody, whereas seropositive results among neighborhood contacts was 5% (n = 2) for the 2010 patient and 23% (n = 12) for the 2012 patient, as well as 2 of 3 IDU contacts for the 2012 case. Factors significantly associated with seropositivity were having skin wounds, sores, or ulcers (odds ratio OR, 4.6; 95% confidence interval CI, 1.2–17.8) and IDU (OR, 18.0; 95% CI, 1.6–194.0). Burkholderia pseudomallei was isolated from soil collected in the neighborhood of the 2012 patient. Conclusions. Taken together, isolation of B. pseudomallei from a soil sample and high seropositivity among patient contacts suggest at least regional endemicity of melioidosis in Puerto Rico. Increased awareness of melioidosis is needed to enable early case identification and early initiation of appropriate antimicrobial therapy.