Background Acute lymphoblastic leukemia (ALL) is the most common cancer diagnosed in children worldwide. This study was conducted to find out the trends in the bacteriological spectrum and antibiotic susceptibility on blood culture in newly diagnosed children with acute lymphoblastic leukemia during the induction phase at a pediatric oncology unit in South Punjab, Pakistan. Methodology This cross-sectional study was conducted from January 1, 2020, to June 30, 2021. A total of 263 newly diagnosed ALL cases of both genders aged up to 16 years were included. Adopting full aseptic measures, the blood samples of all children were sent for culture and sensitivity testing to the institutional laboratory immediately after collection on the eighth day of the induction phase in all children. Bacterial isolates and their sensitivity/resistance patterns were noted. Results Out of 263 children with ALL, 172 (65.4%) were males. Overall, the mean age was 7.4±3.4 years (ranging between 1 and 16 years). B-cell type was the commonest type noted in 204 (77.6%) children. Out of a total of 52 cases with positive blood culture findings for bacterial isolates, there were 28 (53.8%) cases with gram-negative bacterial isolates, while 24 (46.2%) were gram-positive bacterial isolates. Escherichia coli (E. coli) was the commonest type of gram-negative bacteria noted among 18/28 (64.3%) cases, while Staphylococcus aureus (S. aureus) was the most frequent gram-positive bacterial isolates in 13/24 (54.2%). We found meropenem, linezolid, clindamycin, piperacillin, tazobactam, and amikacin to have the highest antimicrobial sensitivities, while commonly adopted antibiotics such as ciprofloxacin, cefotaxime, cefoperazone, amoxiclav, and ampicillin were found to have high resistance rates. Conclusion Gram-negative bacterial isolates formed the majority of the positive blood culture cases. Escherichia coli , Staphylococcus aureus , and Klebsiella pneumonia (K. pneumonia) were the most common types of bacterial isolates. Routinely used antibiotics such as ciprofloxacin, cefotaxime, cefoperazone, and ampicillin were found to have high rates of resistance.