Created attachment 264
macro generating the issue with proof of it

To fix this problem, we need to be able to reproduce it. To reproduce it, we need a runnable macro and a folder of test images, uploaded as a ZIP archive.

Created attachment 265
runnable macro

thanks Wayne. any image should work, cannot attach mine because they are too big

I tried RGB images and they do not work. Please upload a folder of test images that do work with the macro.

Created attachment 266
offending image stack

if you run the macro on the folder containing this image stack you can reproduce the issue

Here is a version of your macro that should work better. I marked the changes with "//**".

dir = getDirectory("Choose Sorce Directory"); //with slash at the end
dir2 = getDirectory("Choose Destination Directory");
acq = getFileList(dir);
setBatchMode(true); 
for (i=0; i<acq.length; i++) {
	if (endsWith(acq[i], "tif")) {
		open(dir+acq[i]);
		run("Make Composite");
		run("Split Channels");
		close("C3-"+acq[i]);
		imageCalculator("Subtract stack", "C2-"+acq[i], "C1-"+acq[i]);
		//Save Z-projection of 169
		selectWindow("C2-"+acq[i]);
		run("Despeckle", "stack");   //**
		run("Z Project...", "projection=[Average Intensity]");
		selectWindow("AVG_C2-"+acq[i]);
		path = dir2+"Zproj-169-BGsubtr-"+acq[i]; //**
		run("Save", "save=&path"); //**
		close("AVG_C2-"+acq[i]);
		//Save Z-projection of SHG
		selectWindow("C4-"+acq[i]);
		run("Despeckle", "stack"); //**
		run("Z Project...", "projection=[Average Intensity]");
		selectWindow("AVG_C4-"+acq[i]);
		path = dir2+"Zproj-SHG-BGsubtr-"+acq[i]; //**
		run("Save", "save=&path"); //**
		close("*");
	}
}

thanks Wayne.  I reported the bug because my code used to work but now something changed with latest version. Best

You are probably using stacks for the first time. "run('Despeckle')" displays a dialog if the active image is a stack. "run('Despeckle','stack')" never displays a dialog.