Microbial species often occur in complex communities and exhibit intricate synergistic and antagonistic interactions. To avoid predation and compete for favorable niches, bacteria have evolved specialized protein secretion systems. The type VI secretion system (T6SS) is a versatile secretion system widely distributed among Gram-negative bacteria that translocates effectors into target cells or the extracellular milieu via various physiological processes. Pseudomonas aeruginosa is an opportunistic pathogen responsible for many diseases, and it has three independent T6SSs (H1-, H2-, and H3-T6SS). In this study, we found that the H3-T6SS of highly virulent P. aeruginosa PA14 is negatively regulated by OxyR and OmpR, which are global regulatory proteins of bacterial oxidative and acid stress. In addition, we identified a H3-T6SS effector PA14_33970, which is located upstream of VgrG3. PA14_33970 interacted directly with VgrG3 and translocated into host cells. Moreover, we found that H3-T6SS and PA14_33970 play crucial roles in oxidative, acid, and osmotic stress resistance, as well as in motility and biofilm formation. PA14_33970 was identified as a new T6SS effector promoting biofilm formation and thus named TepB. Furthermore, we found that TepB contributes to the virulence of P. aeruginosa PA14 toward Caenorhabditis elegans. Overall, our study indicates that H3-T6SS and its biofilm-promoting effector TepB are regulated by OxyR and OmpR, both of which are important for adaptation of P. aeruginosa PA14 to multiple stressors, providing insights into the regulatory mechanisms and roles of T6SSs in P. aeruginosa.