Yes. An E-2 spouse is employment-authorized incident to status and may work in the enterprise or anywhere else. His wages count toward the clinic's payroll but not toward the marginality test.
Spousal work is permitted; it does not cure marginality
Hypothetical example: a Quispamsis physiotherapist plans to have her husband manage reception and billing at the U.S. clinic. Once admitted in E-2 derivative status, he is employment-authorized incident to that status, so he may take that job or a different one.
What his employment does not do is make the enterprise non-marginal, because the test looks at whether the business generates more than a living for the investor and her family, or will create jobs for others. The staffing plan should therefore show employees outside the household, whether the second therapist already on payroll or hires projected within a realistic period. Children under 21 may accompany and study but may not work.
If the couple's long-term intention is permanent residence, they should be told plainly that E-2 provides no direct path to it, and that a separate route would be needed.