Background Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection, gonorrhoea, is known to form biofilms rich in extracellular DNA on human cervical cells. Biofilm formation is conducive to increased antimicrobial resistance and evasion of the host immune system, potentially causing asymptomatic infections. Using plate-based assays we have previously shown that disruption of a potential extracellular DNA ligase, Lig E, in N. gonorrhoeae (Ngo-Lig E) impacts biofilm formation. In this research, we further explored this phenotype using confocal and scanning electron microscopy to directly visualise the morphology of microcolony and biofilm formation. Biofilm growth on artificial surfaces and on 3-dimensional human vaginal epithelial tissue was evaluated for strains where ngo-lig E was either disrupted or overexpressed. Results Results demonstrated that Ngo-Lig E was important for the formation of robust, compact N. gonorrhoeae microcolonies, as well as extensive biofilms on artificial surfaces. The ngo-lig E deletion strain also had the highest tendency to be retained on the surface of epithelial tissues, with decreased invasion and damage to host cell layers. Conclusions These findings support a role for Ngo-Lig E to be secreted from N. gonorrhoeae cells for the purpose of inter-cell adhesion and biofilm formation. We suggest that Ngo-Lig E strengthens the extracellular matrix and hence microcolony and biofilm formation of N. gonorrhoeae by ligation of extracellular DNA.