Biological events—including outbreaks and pandemics, biological weapons use, or accidental laboratory release—have the potential to be extremely disruptive. The ability to accurately investigate, identify origins of, and attribute these events is critical for deterring deliberate events and implementing interventions to prevent future natural or accidental events. However, historical examples of biological event attribution and origins investigations illustrate significant gaps in processes, from technical capabilities to communications, and have lacked conclusive consensus among decision makers, the public, and scientists. This study aimed to assess attitudes and expectations of a broad range of stakeholders regarding investigations and evidence generated for biological attribution. We interviewed 41 experts in disciplines related to attribution and investigations and analyzed interview content using a mixed-methods approach. Interviews generated a list of factors to consider when planning or conducting investigations, presented here. Opinions concerning the conduct and reporting of biological samples analyses and perceptions of feasibility of attribution varied among interviewees representing different fields of study. Participant opinions varied less in regard to requirements, protocols, and guidelines thought to be important to maintain confidence and trust in an investigation and evidence. Findings from this study can inform planning for future events.