In general, yes. It's just a rubber seal, but you'd expect about the same degree of barrier quality as from rubber or polymer gloves, microfuge tubes, etc. The whole point of the chamber slides is to keep the chambers separated, and people use them routinely to examine various nutrient conditions, for example, where an amino acid or salt is in one well but not another.

Now, your small molecule might well be permeable through the barrier, but that's something you'd have to specifically test. Most molecules, however, are going to stay in the well you put them in. I'd be much more worried about vaporization and diffusion across the wells by that mechanism, myself - and even that's only a very, very tiny worry for the average small molecule.

Dear Neal, I don't use Lab-Tek-II chambers but have experience with Millicell EZ slides (4-well glass). I have used them to test molecular sensors for small analytes (L-cys, HS-/H2S for example) as well as gaseous molecules like CO and have never seen leaks into next door wells even after 6 h of incubation time. I have also used them in the case of CO-releasing molecules and haven't seen leak as well. Maybe this information will be of help for your experiments.