Introduction Medical professionals use social media to interact with other healthcare professionals, discuss medical issues and promote healthcare information. These platforms have tremendous power to promote healthcare messages but also have potential to damage the profession if used inappropriately. It is currently unknown how others perceive medical doctors’ Twitter activity and, therefore, we conducted an online survey exploring these views. Methods We used a Google Forms questionnaire consisting of 21 questions, which we distributed on Twitter, exploring doctors’, patients’, the public's and other healthcare professionals’ views of doctors’ Twitter activities. We investigated factors that were associated with mistrust by univariate and multivariate analysis. Results Seven-hundred and twenty-six respondents completed the survey. By univariate analysis, a higher proportion of non-doctors reported witnessing unprofessional behaviour and potential breaches of patient confidentiality compared with doctors (p<0.01). In addition, a significantly higher proportion of non-doctors felt that doctors’ Twitter accounts should be monitored by both their employer and regulator when compared with doctors. By multivariate analysis, the main predictor of mistrust in the profession were those that had previously witnessed unprofessional behaviour (odds ratio 2.70; 95% confidence interval 2.08–3.33; p<0.01). Conclusion There are discrepancies in how doctors and non-doctors view Twitter activity and significant mistrust in the profession was brought about by doctors’ Twitter activity. To help limit this, adherence to current guidelines set out by the General Medical Council and British Medical Association is vital and doctors should be cautious about how their Twitter activity is professionally perceived by others before posting.