Studies of microorganisms are often hindered by a lack of effective genetic tools. One such example is Fusobacterium nucleatum, a gram-negative anaerobe associated with various human infections, including those causing periodontal disease and preterm birth. The first double-crossover allelic-exchange mutant in F. nucleatum was recently constructed using sonoporation, a novel ultrasound-mediated intracellular delivery method, demonstrating potential for bacterial gene transfection. To better unveil its mechanism, the current study examines the factors affecting the outcome of sonoporation. Delivery of Texas Red-conjugated dextran into F. nucleatum by sonoporation was at least twice as efficient as that by electroporation, and sonoporation was nonbactericidal, unlike electroporation. The delivery efficiency was affected by the acoustic pressure amplitude, the duty cycle, and the quantity of microbubbles used to initiate cavitation but not by the pulse repetition frequency of ultrasound application. To examine the involvement of homologous recombination in sonoporation-mediated mutant construction, the highly conserved recA gene, which carried most of the consensus residues, including the P loop, was identified in F. nucleatum, and a double-crossover recA mutant of F. nucleatum 12230, US1610, was constructed by sonoporation. The mutant exhibited increased sensitivity to UV exposure compared with that of the wild type, indicating that the RecA function in F. nucleatum was conserved. Interestingly, US1610 was also sensitive to ultrasound treatment, suggesting the likely involvement of RecA in postsonoporation repair and survival. Since sonoporation has consistently generated one-step double-crossover mutants in F. nucleatum by use of intact suicide plasmids, this technology may be developed into an efficient tool for streamlining mutant construction in bacteria.