Various challenges significantly impact the transformation process, both directly and indirectly. One major concern is the overgrowth of Agrobacterium, which can occur after the co-cultivation phase of the explant. This issue has been observed in several plant species and can disrupt the entire transformation process. To address this problem, multiple strategies are being employed to prevent the unwanted bacterial growth. Once overgrowth occurs, it becomes extremely difficult to rectify, making prevention crucial. Several factors play a role in regulating this phenomenon, including the nature of the explant, the A. tumefaciens strain, T-DNA vector, co-cultivation conditions (time and environment), the use of acetosyringone, washing medium, and antibiotics (type, concentration, combination, and incubation period). In this article, we review these factors based on available research reports.