In this investigation, we sought to investigate the characteristics of Citrobacter spp. infections. A retrospective cohort study in a 700-bed, tertiary care, university hospital was carried out during the period from June 1994 to January 2006. Seventy-eight patients (70 adults) with Citrobacter spp. isolates were identified. C. freundii was more common (71.8%), followed by C. koseri (23.1%) and C. braakii (3.8%). The most common associated clinical syndromes were urinary tract infections (52.6% of cases, including eight cases of asymptomatic bacteriuria), as well as intra-abdominal (14.1%), surgical site (7.7%), skin and soft tissue (6.4%), and respiratory tract infections (6.4%). In 29.5% of patients, Citrobacter spp. isolates were associated to polymicrobial infections, principally at sites other than the urinary tract. Antibiograms of 38 consecutive Citrobacter spp. isolates (29 C. freundii) were available. Most active agents were colistin (100%), fosfomycin (100%), imipenem (97.4%), gentamicin (89.5%), nitrofurantoin (89.5%), ciprofloxacin (80.6%), and cefepime (73.7%). Most patients (82.1%) had at least one underlying illness. Combination antimicrobial therapy was administered in 28.2% of cases. One patient died during hospitalization. The length of hospital stay was longer in patients with polymicrobial compared to monomicrobial infections (23 versus 13 days, respectively, p = 0.02). The isolation of Citrobacter species, although rather infrequent, was clinically relevant in the great majority of cases. Further attention should be paid to these pathogens.