The presence of humans within the natural environment is increasing worldwide. Assessing the impact of such activities on wildlife is crucial for declining populations where human disturbance adds to existing pressures. Here, we investigated how human activities at night influenced Little Penguin Eudyptula minor numbers and behaviours (specifically return time, number of vocalizations and time spent in vigilance) on Granite Island, a declining population in South Australia, Australia. We combined data from regular night surveys with continuous video and audio monitoring to assess the impact of human activities on the Little Penguins. The use of white light (i.e. from torches or camera flashes) by people was the most frequent activity recorded at night (recorded on 65% of the monitored nights). Fewer penguins were found on land at night when Dogs Canis lupus familiaris were present, but not when the number of people increased, when concerts occurred, or when white lights were used. Little Penguins were observed more often returning late from sea at night when Dogs were present and when white lights were used, but not when concerts occurred. An increase in penguin vocalizations at night correlated with the presence of Dogs and the occurrence of concerts, whereas penguins vocalized less when white lights were used. The time Little Penguins spent in vigilance did not correlate with any of the disturbances analysed. Our study also highlights the impact of coronavirus disease 2019 (COVID‐19) on wildlife, as the occurrence of human activities increased significantly following the implementation of the COVID‐19 health protection measures. These results add to a growing body of literature suggesting that human activities on land, and their consequent disturbance(s), may affect the numbers and behaviours of wildlife and that appropriate measures need to be developed to limit such impacts.