Abattoirs generate enormous volume of wastewater, containing high level of pathogenic bacteria, antibiotics and organic matter. Discharge of untreated abattoir wastewater can cause surface and groundwater pollution and negatively affect aquatic ecosystem, thereby, posing risks to public health. This study is aimed at determining antibiotic resistance pattern of the Gram-negative bacteria isolated from abattoir wastewater in Amosun, Ibadan, Nigeria. Wastewater samples were aseptically collected from the abattoir in sterile bottles using standard method and was analyzed for their physicochemical properties. Standard pour plate method was used for bacterial enumeration and isolation using Nutrient agar and MacConkey Agar, respectively. Identification of the isolates was done using conventional methods. Antibiotic susceptibility test was performed against 5 selected antibiotics using the Kirby-Bauer’s disk diffusion technique according to the Clinical and Laboratory Standards Institute guidelines. The mean physicochemical parameters of the wastewater showed a pH of 6.33, turbidity of 250 NTU and a strong odour. The mean total heterotrophic bacteria count was 2.42 x 102 CFU/mL. A total of 30 Gram-negative bacteria including: Pseudomonas spp. (11), Enterobacter spp. (7), Klebsiella spp. (4), Aeromonas spp. (2), Providencia spp. (2), Escherichia coli (1), Proteus sp. (1), Serratia sp. (1), and Alcaligenes sp. (1) were isolated. The antibiotic susceptibility profile showed a total resistance to amoxicillin- clavulanic and high resistance rates: 96.6% to both ceftriaxone and ceftazidime, 73% to imipenem. The antibiotic resistance of diverse potential pathogens in abattoir wastewater suggests a critical need for sustainable wastewater treatment and regulate the use of antibiotics in animal husbandry.