Background: Participation in and tolerability of primary colonoscopy screening are presumed to be relatively low. The present study aimed to test its feasibility in a well-informed population of hospital staff using an intensive information campaign, and to identify factors associated with screening colonoscopy rated as uncomfortable. Methods: Data were collected using standardized forms. Results: Out of 1,090 invited employees (50–65 years), 447 (41.0%) participated. Bowel preparation and colonoscopy were rated as ‘somewhat to very uncomfortable’ by 79.5 and 21.9%, respectively. 96.3% of participants were willing to repeat colonoscopy in the future. Participants rating colonoscopy as uncomfortable were more likely unwilling to repeat the procedure (OR 8.026, CI 2.667–24.154). Multivariate analysis (age- and gender-adjusted) showed an association of colonoscopy rated as uncomfortable with: abdominal pain during colonoscopy (OR 3.185, CI 1.642–6.178), other pain (OR 2.428, CI 1.335–4.416), flatulence (OR 2.175, CI 1.219–3.881), embarrassment (OR 2.843, CI 1.350–5.989), abdominal pain after colonoscopy (OR 1.976, CI 1.041–3.751), and a prolonged procedure time (OR 1.000, CI 1.000–1.001). Conclusions: Acceptance of primary colonoscopy screening for colorectal neoplasia was high, although participants with symptoms during and after colonoscopy were more likely to rate colonoscopy as uncomfortable. This type of opportunistic screening procedure is suitable for the introduction of screening programs and may be useful in areas that have no access to population-based screening.