Nurses make up the largest proportion of the health workforce and their ability to be assertive with high self-esteem is the key to reducing major medical errors and observing early signs of unsafe condition in care delivery. The aim of this study was to investigate the effect of assertiveness training on assertive behaviour and self-esteem of nurse interns in tertiary hospitals in Enugu. The study adopted a quasi-experimental design using two instruments: Rathus assertiveness scale and Rosenberg self esteem scale. A total population of ninety-nine (99) nurse interns participated in the study. Reliability test of the instruments showed (r) of 0.86 for Rosenberg self-esteem Scale, and (r) of 0.92 for Rathus assertiveness scale. Post-test data were collected one week and six (6) weeks after the assertiveness training from the intervention groups. Data were analysed using descriptive and inferential statistics. The findings revealed that the level of assertiveness of the nurse interns was low (Control (C) =22.5, Intervention (I) =19.6), while the level of self-esteem of the nurse interns was moderate (C= 45.5, I=41.1. There was a significant increase in the assertive behavior and self-esteem of the nurse interns six weeks after the intervention (t = 2.35, P = 0.02 at P < 0.05); (t = 2.35, P = 0.02 at P < 0.05) respectively. The level of self-esteem was significantly higher for nurse interns who were assertive than for those who were non-assertive (P-value = .000). Nurse interns in Nigeria currently have relatively low level of assertiveness, however, their level of self-esteem is moderately high.