The broth microdilution method for telavancin susceptibility testing was revised and now utilises DMSO as solvent for stock solution preparation and diluent for stock solution dilution, following CLSI guidelines for water-insoluble agents. The revised method also incorporates polysorbate 80 in the test medium to mitigate drug binding to plastics. This revised methodology provides more accurate and reproducible MIC determinations, which results in values lower than the previously established method. This study was conducted to re-establish telavancin potencies and susceptibility profiles (using updated interpretive criteria) against a collection of uncommon clinical pathogens (3821 isolates). Telavancin showed MIC50 values of 0.06 mg/L against tested staphylococcal species (MIC50/90, 0.03/0.06 mg/L; 98.1–100.0% susceptible), with lower results for Staphylococcus hominis (MIC50, ≤0.015 mg/L), Staphylococcus lugdunensis (MIC50, ≤0.015 mg/L) and Staphylococcus simulans (MIC50, 0.03 mg/L). Vancomycin (MIC50, 1 mg/L), daptomycin (MIC50, 0.12–1 mg/L) and linezolid (MIC50, 0.25–1 mg/L) had MIC50 results at least four-fold higher than telavancin against CoNS. Streptococci (99.2–100.0% susceptible) displayed telavancin MIC50 values of ≤0.015–0.03 mg/L. Vancomycin (MIC50, 0.25–0.5 mg/L) and linezolid (MIC50, 0.5–1 mg/L) had higher MIC50 results against streptococci, whilst daptomycin MIC50 values varied from ≤0.06 mg/L to 0.5 mg/L. Micrococcus, Listeria and Corynebacterium spp. were inhibited by telavancin at ≤0.015, ≤0.03 and ≤0.06 mg/L, respectively. Telavancin exhibited potent in vitro activity against this collection, greater than comparators (daptomycin, linezolid, vancomycin). This study provides new baseline MIC results for telavancin and confirms the spectrum and potency of telavancin against less commonly encountered Gram-positive species.