The objective of this study was to investigate the risks associated with the use of roof-harvested rainwater (RHRW) and the implication of pigeons as the most likely source of contamination by testing for antibiotic resistance profiles of Escherichia coli. A total of 239 E. coli were isolated from thirty fresh pigeon faecal samples (130 isolates), 11 RHRWtanks from three sites in Pretoria (78) and two in Johannesburg (31). E. coli isolates were tested against a panel of 12 antibiotics which included ampicillin, amoxicillin, amikacin, cefoxitin, ceftriaxone, chloramphenicol, ciprofloxacin, cotrimoxazole, enrofloxacin, gentamicin, nalidixic acid and tetracycline. In all samples, resistance to ampicillin (22.7.9 %), gentamicin (23.6 %), amikacin (24 %), tetracycline (17.4) and amoxicillin (16.9 %) were the most frequently encountered form of resistance. However, a relatively higher proportion of isolates from pigeon faeces (67.3 %) were antibiotic resistant than those from RHRW (53.3 %). The highest number of phenotypes was observed for single antibiotics, and no single antibiotic resistance was observed for chloramphenicol, ceftriaxone, gentamicin, cefoxitin, cotrimoxazole, although they were detected in multiple antibiotic resistance (MAR) phenotypes. The highest multiple antibiotic resistance (MAR) phenotypes were observed for a combination of four antibiotics, on isolates from JHB (18.8 %), pigeon faeces (15.2 %) and Pretoria (5.1 %). The most abundant resistance phenotype to four antibiotics, Ak-Gm-Cip-T was dominated by isolates from pigeon faeces (6.8 %) with Pretoria and Johannesburg isolates having low proportions of 1.3 and 3.1 %, respectively. Future studies should target isolates from various environmental settings in which rainwater harvesting is practiced and the characterisation of the antibiotic resistance determinant genes among the isolates.