Drones are increasingly used in marine science for monitoring large megafauna in nearshore areas. Remotely operated, autonomous missions have the potential to improve the overall efficiency of drone-based research. We assessed the utility of autonomous drone operations by comparing real-time detection rates of marine megafauna (i.e., dolphins, rays, sharks, turtles) between a remotely operated Drone-In-A-Box (DIAB) system using pre-programmed missions and standard site-operated manual flight procedures. Megafauna were identified in real time during each mission, and missed detections were quantified through post-analysis of drone footage. Seventy-one missions were completed, with autonomous and manual flights operating concurrently. There were 107 and 117 real-time megafauna observations recorded for autonomous and manual operations, respectively. Post-flight analysis determined an overall missed detection of 52.4% for autonomous and 30.4% for manual operations, with undercounting higher for autonomous operations across all faunal groups. Real-time dolphin detection had the highest agreement with post-flight analysis, while real-time turtle detection proved the most difficult. Overall, remotely operated, autonomous drones have the potential to enhance long-term marine megafauna research, particularly when combined with post-flight analysis. Integrating artificial intelligence into autonomous drone operations will also be beneficial, especially for shark surveillance programs where real-time detection is essential for beach-user safety.