Objectives To comparatively evaluate the antimicrobial activities of colistin and polymyxin B with those of other antimicrobials against a worldwide collection of 40 625 Gram-negative bacilli. Methods Antimicrobial susceptibility testing was performed and interpreted using the CLSI broth microdilution method except for colistin against Enterobacteriaceae. Results The polymyxins showed potent in vitro activities (MIC90, ≤0.5-1 mg/L) against this large collection of clinical isolates, with very low resistance rates (<0.1%-1.5%). Resistance to the polymyxins remained stable among organisms tested except for Klebsiella spp. isolates collected from the Asia-Pacific and Latin American regions, where a trend towards greater resistance was observed (P ≤ 0.05). In addition, an important reduction in imipenem susceptibility among Acinetobacter spp. and Klebsiella spp. was demonstrated in most geographical regions. Conclusions Although the polymyxins showed excellent in vitro activity against the vast majority of Gram-negative bacilli evaluated, a trend to greater resistance was observed in the Asia-Pacific and Latin American regions. Therefore, the clinical use of polymyxins must be cautious and surveillance monitored.