Residues of antimicrobials used in farm can exert selective pressure and accelerate the occurrence of multidrug resistant bacteria in litter. This study aimed to investigate the resistance profile of Escherichia coli isolated from poultry litter. A total of 101 E. coli strains was isolated from 229 litter samples collected and stored for two months in the laboratory at room temperature. Antimicrobial susceptibility testing was performed using the disk diffusion method. An overall resistance prevalence of 58.4% (95% CI: 48.8–68.0) was obtained with 59 E. coli strains resistant to various antimicrobial agents. High levels of resistance were observed with ciprofloxacin (21/59: 36%), imipenem (27/59: 45%), norfloxacin (44/59: 74%), ceftriaxone (44/59: 74%), and levofloxacin (44/59: 75%). These antimicrobials classified under the Watch group by WHO are indicators of the high AMR risk to public health in Cameroon. Multivariable logistic regression analysis revealed that a greater probability of high level of E. coli multidrug resistance was associated with lack of training in poultry farming (OR = 0.13, p = 0.01), less experience in poultry farming (OR = 11.66 p = 0.04), and the high frequency of digestive tract disease (OR = 0.10; p = 0.001). This study revealed that poultry litter constitutes a potential source of dissemination of resistant germs from farm animals to the environment and humans.